The fundamental goal of this project is to define a primary, molecular mechanism of breast cancer, a major disease affecting one in nine women in the U.S. over their lifespan. The model system employed to achieve this objective is an extensive series of human papilloma virus (HPV) DNA- immortalized human mammary epithelial cell lines that have been well characterized for karyotype, for growth factor requirements, and for the single viral oncoprotein, HPV E6, required for their immortalization. The HPV-immortalized and paired normal, senescing cells, from which they are derived, offer a unique opportunity to identify genes responsible for maintenance of the senescence phenotype in mammary epithelial cells. The finding of a consensus loss of chromosome 19 in the HPV cells has led to the hypothesis that this chromosome contains a gene or genes, which when lost or inactivated, allows mammary epithelial cells to escape senescence or terminal differentiation. Key experiments, using microcell hybrids to reconstitute chromosome 19, will test the hypothesis directly. Parallel introduction of other human chromosomes (e.g. 4 and 15) into HPV cells serve as controls for microcell hybridization efficiency and as controls for chromosome specificity in reconstituting the senescent phenotype. To clone the specific gene(s) responsible for escape from senescence, differential screening of a chromosome 19 genomic library as well as subtractive hybridization are proposed. For differential screening, CDNA from normal and HPV cells will be used to screen duplicate filters from a chromosome 19 lamda library. Subtraction between normal CDNA and HPV MRNA will elucidate genes expressed exclusively in the senescing cells. Only the E6 oncoprotein is required for HPV16 immortalization of mammary epithelial cells. As the E6 protein is involved in the reduction of the p53 gene product, a potential tumor suppressor in breast cancer, our mammary cell system will be utilized to study the respective and interactive roles of p53 and E6 in the senescent phenotype. To test whether the continued function of E6 is required for the immortal phenotype, knockout strategies, using antisense oligonucleotides as well as antisense RNA will be employed. Our planned studies offer insights into the disease of breast cancer by the identification of genes necessary to preserve the delicately balanced states of senescence and cellular transformation.